Submicronic particles are particles less than 1 micron and include nano scale particles. Nano particles are part of an emerging science called ‘nano technology’. The word nano technology comes from the Greek prefix ‘nano’ meaning “one billionth”. In modern scientific parlance, a nanometer is one billionth of a meter, about the length often hydrogen atoms placed side by side in a line. The smallest things that an unaided human eye can see are 10,000 nanometers across. Nano particles are typically and generally spherical in shape.
Nanoscience, simply, is the study of the fundamental principles of structures with at least one dimension roughly between 1 and 100 nanometers and Nanotechnology is the application of these nanostructures into useful nanoscale devices.
Nano scale particles of substances exhibit properties unlike the properties of their macro counterparts often with stunning new results. Nano scale is unique because it is the size scale where the familiar day-to-day properties of materials like conductivity, hardness or melting point meet the more exotic properties of the atomic and molecular world such as wave-particle duality and quantum effects. At the nano scale, the most fundamental properties of the materials and machines depend on their size in a way they don't at any other scale. For e.g. a nano scale wire or circuit component does not necessarily obey Ohm's law. Nano-scale particles have unique physical properties (e.g. optical, dielectric, magnetic, mechanical), transport properties (e.g., thermal, atomic diffusion) and processing characteristics (e.g., faster sintering kinetics, super-plastic forming).
Physicist Richard Feynman first described the possibility of molecular engineering. In 1959 Feynman gave a lecture at the California Institute of Technology called “There's Plenty of Room at the Bottom” where he observed that the principles of physics do not deny the possibility of manipulating things atom by atom. He suggested using small machines to make even tinier machines, and so on down to the atomic level itself. Nano technology as it is understood now though, is the brainchild of Feynman's one-time student K. Eric Drexler. Drexler presented his key ideas in a paper on molecular engineering published in 1981, and expanded these in his books Engines of Creation and Nano systems: Molecular Machinery, Manufacturing and Computation, which describes the principles and mechanisms of molecular nano technology.
In 1981 the invention of the Scanning Tunneling Microscope or STM, by Gerd Binnig and Heinrich Rohrer at IBM's Zurich Research Labs, and the Atomic Force Microscope (AFM) five years later, made it possible to not only take photos of individual atoms, but to actual move a single atom around. Soon after, John Foster of IBM Almaden labs was able to spell “IBM” out of 35 xenon atoms on a nickel surface, using a scanning tunneling microscope to push the atoms into place.
A nanometer is a magical point on the dimensional scale. Nano structures are at the confluence of the smallest of Human-made devices and the largest molecules of the living things. Nano technology exploits the new physical, chemical and biological properties of systems that are intermediate in size, between isolated atoms/molecules and bulk materials, where the transitional properties between the two limits can be controlled.
The synthesis and characterization of nano particles has received attention in recent years because of the possibility of their widespread use in industry and chemistry. Nanotechnology is gaining importance in areas such as biomedical sciences, optics, electronics, magnetics, mechanics, ceramics, catalysis and energy science. However, the preparation of such nano structured materials poses several unique challenges. A range of nano particles has been produced by physical, chemical and biological methods.
Two approaches have been adopted for nano fabrication—The Top down processes, which include the methods of synthesis that carve out or add aggregates of molecules to a surface. The second is the bottom up approach, which assembles atoms or molecules into nano structures.
PHYSICAL methods include Electron beam lithography, Scanning probe method, Soft lithography, Microcontact printing, Micromoulding.
In Electron Beam Lithography, an electron beam scans the surface of a semiconductor containing a buried layer of quantum well material. The resist gets removed where the beam has drawn a pattern.
Soft lithography is an extension of the previous technique and overcomes the impracticability of applying electron beam lithography to large scale manufacturing by making a mould or a stamp, which can be used repeatedly to produce nanostructures. In Micro contact printing, the PDMS stamp is inked with a solution consisting of organic molecules called thiols and then pressed against a thin film of gold on a silicon plate. The thiols form a self-assembled monolayer on the gold surface that reproduces the stamp pattern; features in the pattern can be as small as 50 nm. In Micromoulding, the PDMS stamp is placed on a hard surface, and a liquid polymer flows into the recesses between the surface and the stamp. The polymer solidifies into the desired pattern, which may contain features smaller than 10 nm.
Scanning probe microscope can image the surface of conducting materials with atomic scale detail. Hence single atoms can be placed at selected positions and structures can be built to a particular pattern atom by atom. It can also be used to make scratches on a surface and if the current flowing from the tip of the STM is increased the microscope becomes a very small source for an electron beam which can be used to write nanometer scale patterns. The STM tip can also push individual atoms around on a surface to build rings and wires that are only one atom wide.
In Sonochemical method, an acoustic cavitation process is used to generate a transient localized hot zone with extremely high temperature gradient and pressure (Suslick et al. 1996). Such sudden changes in temperature and pressure bring about the destruction of the sonochemical precursor (e.g., organometallic solution) and the formation of nanoparticles.
Hydrodynamic cavitation consists of synthesis of Nanoparticles by creation and release of gas bubbles inside sol-gel solutions (Sunstrom et al. 1996).
High energy ball milling is already a commercial technology, but has been considered dirty because of contamination problems from ball-milling processes. However, the availability of tungsten carbide components and the use of inert atmosphere and/or high vacuum processes have reduced impurities to acceptable levels for many industrial applications. Common drawbacks include the low surface area, the highly polydisperse size distributions, and the partially amorphous state of the as prepared powders.
CHEMICAL methods include Wet chemical preparation, Surface passivation, Core shell synthesis, Organometallic precursor, Sol gel method, Langmuir-Blodgett method, Precipitation in structured media, Zeolites, Micelles and inverse micelles formation.
A number of chemical strategies are now available for the construction of higher order structures. Organic molecules can be linked together by molecular recognition. For example, synergistic noncovalent donor acceptor interactions can give rise to intertwined rings (catenanes). Liquid crystal polymers having self-organized structures can be formed from organic molecules containing head groups capable of complementary hydrogen bonding interactions. Organic molecules can be assembled around metal ions such as Cu(I) that provide stereo chemical constricts in the construction of double helices. The synthesis of inorganic clusters, by contrast, is usually dependent on passivating the surface of a growing aggregate by capping the surface sites with stabilizing ligands.
Wet Chemical Preparation method involves the reaction between a metal ion and the desired anion under controlled conditions to generate nanocrystals of desired size.
BIOLOGICAL methods include Biomineralization using Bacteria, Yeast, Fungi, Plants and Biotemplating using Ferritin, Lumazine synthase, Virus Surface layers DNA etc.
A few attempts have been made to synthesize sulfides, typically cadmium sulfide (CdS) using microorganisms. It was shown that CdS nanoparticles can be synthesized in the yeasts Candida glabrata and Schizosaccharomyces pombe. These nanoparticles are coated with short peptides known as phytochelatins, which have the general structure (y-Glu-Cys)n-Gly where n varies from 2-6. The nanoparticles are size reproducible, more monodisperse, and have greater stability than synthetically produced nanoparticles. Further work on microbial synthesis of CdS nanoparticles is scant and is limited to studies on characterization and efficient production in batch cultivation.
U.S. Pat. Nos. 5,876,480 & 6,054,495 describe a process for creating unagglomerated metal nano-particles, comprising the steps of                (a) forming a dispersion in an aqueous or polar solvent, the dispersion including unpolymerized lipid vesicles, the unpolymerized lipid vesicles each comprising at least one lipid bilayer, the lipid bilayer including a negatively charged lipid that has an anionic binding group, and the lipid vesicles having catalytic first metal ions bound thereto by ionic bonding,        (b) combining the dispersion of step (a) with a metallization bath containing free second metal ions to form a mixture, and        (c) incubating the mixture of step (b) at a temperature sufficient to reduce said free second metal ions and to form unagglomerated metal nano-particles having an average diameter between about 1-100 nm.        
U.S. Pat. No. 6,068,800 describes a process and apparatus for producing nano-scale particles using the interaction between a laser beam and a liquid precursor solution using either a solid substrate or a plasma during the laser-liquid interaction.
U.S. Pat. Nos. 5,618,475 and 5,665,277 relate to production of particulates having nanoparticle dimensions, such as about 100 nanometers diameter or less, and, more particularly, to apparatus and method for producing nanoparticles of metals, alloys, intermetallics, ceramics, and other materials by quench condensation of a high temperature vapor generated by an evaporator having features effective to isolate evaporation conditions from downstream conditions and to concurrently evaporate materials of dissimilar vapor pressures.
U.S. Pat. No. 5,736,073 describes a process of production of nanometer particles by directed vapor deposition of electron beam evaporant onto a substrate.
U.S. Pat. No. 6,706,902 The continuous process according to the invention includes impregnating support materials and, after thermal activation, drying the support materials by spraying or by fluidized bed technology leads to form precious metal-containing support compositions that are active in the catalysis of oxidation reactions.
U.S. Pat. No. 6,562,403 is broadly concerned with chemical methods of forming ligated nanoparticle colloidal dispersions and recovered ligated nanoparticles which may be in superlattice form.
U.S. Pat. No. 5,698,483 A process for producing nano size powders comprising the steps of mixing an aqueous continuous phase comprising at least one metal cation salt with a hydrophilic organic polymeric disperse phase, forming a metal cation salt/polymer gel, and heat treating the gel at a temperature sufficient to drive off water and organics within the gel, leaving as a residue a nanometer particle-size powder.
The main disadvantages of these methods are that they are expensive and technically difficult and too slow for mass production. Most of the techniques are also capital intensive as well as inefficient in materials and energy use. The known methods are difficult to control in order to acquire a desired size and shape of the nano-scale particles to be produced. Many of these synthesis techniques also require the use of a vacuum unit and involve environmental concerns about chemical waste disposal. Almost all of the methods used in the manufacture of submicronic scale particles use at least one toxic or questionable chemical reagent at any of the three main components of synthesis which are solvent medium used in the synthesis, the reducing agent used in the synthesis and the material used for stabilization. Most of the reported methods rely heavily on the use organic solvents. Particles made with the help of these solvents are not biocompatible and therefore unsuitable for use in biological applications such as biosensors and markers. Many of the reducing agents such the borohydrides, dimethylformamide, hydrazines are very highly reactive chemicals and are hazardous both environmentally and biologically. In some cases isolation and recovery of the particles is difficult for example in the case of surfactants which have an affinity for carbon dioxide. The most significant set of problems arise however in the step of stabilization. Almost all the stabilizing or capping agents used in the form of polymers or chemicals pose hazardous and risky steps either in the final stabilized product or in the process of making the stabilizing substance or in the process of stabilization.
Physical and chemical methods in the manufacture of nanoparticles involve controlling crystallite size by restraining the reaction environment. However, problems occur with general instability of the product and in achieving monodisperse size. The dispersion of nano particles usually display very intense color due to plasmon resonance absorption, which can be attributed to the collective oscillation of conduction electrons, induced by the presence of an electromagnetic field.
Other problem areas in the above mentioned methods are uniform distribution of particles, morphology and crystallinity, particle agglomeration during and after synthesis and separation of these particles from the reactant.
Nano particles are extremely reactive as the coordination of surface atoms in nano particle is incomplete, and can lead to particle agglomeration to minimize total surface or interfacial energy of the system. This problem is overcome by passivating the bare surface atoms with protecting groups. Capping or passivating the particle not only prevents agglomeration, it also protects the particles from its surrounding environment, and provides electronic stabilization to the surface. The capping agent usually takes the form of a Lewis base compound covalently bound to surface metal atoms.
Chemical techniques have therefore been developed to passivate or stabilize these nano particles. It is desirable that nano particles are protected from the environment but are still allowed to maintain their intrinsic properties. It has been shown that the size, morphology, stability and properties (chemical and physical) of these nanoparticles have strong dependence on the specificity of the preparation method and experimental conditions.
The stabilizing of nano particles in a sub micronic regime requires an agent that can bind to the cluster surface and thereby uncontrolled growth or agglomeration of the cluster or discrete particles into larger particles is prevented. The simplest method involves the use of a solvent that acts as a stabilizer of the small clusters. Unagglomerated nano particles can also be made by the use of polymeric surfactants ands stabilizers added to a reaction designed to precipitate a bulk material. The polymer attaches to the surface of the growing clusters and either by steric or electrostatic repulsion prevents further growth of the nano clusters. Commonly used chemical stabilizers include sodium polyphosphate and anionic agents such as thiolates.
Most capping reactions involve additional steps and the capping agents are generally toxic substances.
It is an object of the present invention to provide a stabilizing solution that successfully retains the intrinsic physical and chemical properties of the individual submicronic and particularly nano scale particles molecules.
Another object of this invention is to provide an inexpensive and environmental friendly process for manufacturing a stabilizing solution and to an efficient and ‘green’ process for the stabilizing of submicronic particles.
According to this invention therefore there is provided a sub micronic particle stabilizing solution comprising an aqueous extract of macerated biological cells having pH of 5.5 to 7.5, open circuit potential between +0.02 to +0.2 volt, temperature between 20 degrees to 30 degrees Celsius and concentration of total organic carbon being at least 18,000 ppm.
Typically, the biological cells are plant cells of plant tissue selected from a group of tissues comprising living tissue of leaves, fruits, stems, roots and flowers and parts thereof.
Alternatively, the biological cells are animal cells of animal tissue selected from a group of tissues consisting of tissues of worms, insects, fishes, mollusks, crustaceans, and higher animals.
Still alternatively, the cells are microbial cells selected from a group of microbes, which include bacteria, fungi, yeasts, viruses, protozoa and algae.
In accordance with another aspect of this invention there is provided a method of making a sub-micronic particle stabilizing solution which comprises the steps of    {a} obtaining fresh biological tissue;    {b} macerating the biological tissue in water to form a suspension containing the extracts of the biological tissue;    {c} removing from the suspension suspended particles greater than one micron to obtain a clear concentrated extract;    {d} diluting the concentrated extract with deionized water in a ratio ranging from the original to 10 dilution;    {e} adjusting the temperature to 25 degrees Celsius;    (f) adjusting the pH of the diluted extract to between 5.5 to 7.5 pH;    {g} measuring the open circuit potential to ensure that that potential lies within the range of +0.02 to +0.2 volt; and    {h} measuring the total organic carbon content to ensure that the content is at least 18,000 parts per million in solution.
The biological tissue is typically, macerated by at least one method from a group of macerating methods which consists of grinding, blending, milling, microwave treatment, ultrasonication, sonication, pounding, pressure extrusion, freezing-thawing, irradiation, heat treatment, osmolysis, enzymatic lysis, chemical lysis, vacuum lysis, and differential pressure lysis.
The removal of suspended particles is achieved by filtering the suspension through a sub micronic filter element.
In accordance with a preferable embodiment of the invention, the aqueous extract is treated with a non polar solvent, such as n-cyclohexane for beneficiation of the biomolecules in the extract.
In accordance with still another aspect of this invention there is provided a method of stabilizing sub micronic particles which comprises the steps of dispersing sub micronic particles in the stabilizing solution in accordance with this invention to obtain a resultant in which the concentration of the particles ranges from 5 to 300 ppm; and mixing the resultant for a period of 30 minutes to three hours to obtain a suspension of stabilized solid sub micronic particles.
In accordance with yet another aspect of this invention there is provided a method of stabilizing sub micronic metal particles, during their synthesis, which comprises the steps of dispersing salt of the metal in deionized water to form a solution; adding the formed solution to the stabilizing solution in accordance with this invention to obtain a resultant in which the concentration of the metal is in the range from 5 to 300 ppm and the effective dilution of the stabilizing solution is in the range of 1:1 to 1:10; adding a reducing agent to the resultant; and mixing the resultant for a period of 30 minutes to three hours to obtain a suspension of stabilized solid submicronic particles.
Typically, the sub micronic particles are particles selected from a group of particles from transition metals, alkali metals, alkaline earth metals, rare earth metals, metalloids, a combination of metals, metallic compounds and the sub micronic particles are nano particles and the stabilizing solution is added during the synthesis of the nano particles by a process from a group of process which includes a chemical process, a physical process and a biological process or after the synthesis.
In accordance with one embodiment of this invention the sub micronic particles are silver ions and the step includes dispersing a silver salt in deionized water having conductivity of less than 3 micro siemens.
In accordance with another embodiment of the invention, the sub micronic particles are gold ions and the step includes dispersing a gold salt in deionized water.
Typically, the reducing agent is at least one reducing agent selected from a group containing the stabilizing solution, citric acid, borohydride, sodium sulfide sodium acetate.
The theoretical considerations underlying the invention are as follows:
In nature, the chemical composition of cells across flora and fauna are remarkably similar. Thus living plants cells are very similar chemically to animal cells and microbial cells.
All cells contain biomolecules such as polysaccharides, proteins, lipids and nucleic acids which are made up of building blocks such as monosaccharides, amino acids, fatty acids, nucleotides. In addition many dynamically acting molecules such glutathione, cytochromes, ubiquinone, NADH, FADH, pyruvic acid, citric acid, maleic acids, glycerol are also present. These biomolecules have various reactive groups such as sulfhydryl, amino, imino, carboxyl, hydroxyl, and the like.
When living cells are macerated in water the biomolecules are released in the water. These biomolecules collectively contain all the reactive groups where the elements are in specific proportion to each other. It has been found that these biomolecules have the surprising collective ability to not only reduce metal ions such as gold and silver but also to act sterically on metal nano particles so as to stabilize them either in the process of their synthesis or in a post synthesis process. The binding interaction between biomolecules is relatively weak as compared with the interaction between these particles and typical chemical capping agents such thiols.
Metal nanoclusters are optically transparent and act as dipoles. Conduction and valence bands of metal nanoclusters lie closely and electron movement occurs quite freely. The potential applications of these systems are mainly associated with unusual dependence of the optical and electronic properties on the particle size. Silver particles having 5-50 nm sizes show a sharp absorption band in the 410-420 nm regions. While the same phenomenon with gold nano particles are observed at 520-550 nm.
The size of metal particles prepared by the method described in accordance with this invention depend on the concentration of the metal ions and concentration of biomolecules.
The synthesis of a particle, requires a two-step process, i.e., nucleation followed by successive growth of particles. In accordance with the present invention where the stabilization solution is added during the synthesis in the first step, part of metal ions in solution gets adsorbed on free nucleophilic groups (—SH, OH, NH2) present on the surface of biomolecules in the stabilizing solution and get reduced. The reduced metal atoms thus created act as nucleation centers and facilitate further reduction of metal ions present in the bulk solution. The atomic coalescence leads to the formation of metal clusters and can be controlled by the natural ligands and surfactants forming part of biomolecular mass. Thus biomolecules act as both reducing as well as stabilizing agent. It has been found that a threshold concentration value of biomolecular mass is required to seed the process of submicronic formation and stabilization. This is expressed in terms of the total organic carbon content of the biomolecular mass.
It has also been found by experimentation that the reduction potential plays an important and crucial part in the formation and stabilization of nano particles, particularly, gold and silver nano particles. The electromotive force exhibited by 1 M concentration of a reducing agent and its oxidized form at 25° C. and pH 7.0 is called its standard reduction potential. It is a measure of the relative tendency of the reducing agent to lose electrons.
The reduction potential is measured in positive or negative volts on a scale in which the positive sign denotes a lower reduction potential than the negative sign. Therefore a substance with a standard reduction potential of +0.1 volt has a higher reduction potential than a substance with a standard reduction potential of +0.2 volts and therefore substance with a standard reduction potential of +0.1 volts will reduce the substance having a reduction potential of +0.2 volts. The standard reduction potentials of some of the biomolecules, such as NADH, ubiquinone, cytochrome bk are −0.32 Volt, −0.05 Volt and +0.03 Volt. On the other hand, the standard reduction potentials for conversion of ions such as gold and silver to their solid state are +1.5 Volt and +0.8 Volt, respectively. Thus the findings in accordance with this invention is that many biomolecules which have an effective standard reduction potential higher than that required for the conversion gold and silver ions to solid state particles, can effectively reduce these ions in solution. It is not possible to measure the standard reduction potential of the solutions formed in this invention because the molar concentrations of individual components of the biomolecular mass is unknown. However, dynamically the open circuit potential of the solution can be determined easily. At specific molar concentrations the open circuit potential has a direct relationship with the standard reduction potential. The open circuit potential indicates the initial empirical redox state of the solution.
For the process of our invention, it is critical that the stabilizing solution has an open circuit potential +0.02 to +0.2 Volt, a pH between 5.5 and 7.5 and temperatures between 20 to 30 degrees Celsius are also important parameters. The total organic carbon content of the stabilizing solution must also be at least 18,000 ppm. Purity of water having conductivity less than 3 micro siemens is also of significant importance in optimizing the process of particle formation. Concentration of metal ions in the reacting solution should lie between 5 to 300 ppm; metal ions in the mother solution being 150 to 60000 ppm.
It has been found in the course of experimentation that bio molecules normally found in plant cells, animal and microbial cells in the intact plant, animal or microbe have a reducing potential and are their reducing ability very slowly decreases when exposed to air under ambient conditions and rapidly to denaturing treatment. The reducing ability is therefore inversely related to the freshness of the tissue. It has also been found that the reducing ability varies from tissue to tissue and is inversely proportional to the duration of exposure to air eventually tending to zero.